阅读说明：本技术 一种鹿茸多肽的制备方法及其应用 (Preparation method and application of pilose antler polypeptide ) 是由 丁传波 刘文丛 赵婷 王慈 郑毅男 沈立国 时清评 李沈琦 于 2020-05-27 设计创作，主要内容包括：本发明提供一种鹿茸提取物的制备方法,具体涉及一种鹿茸多肽的制备方法,其特征是,将鲜鹿茸处理后,均质处理,加入碱性蛋白酶后在一定压力作用下进行酶解,酶解结束后对酶解液短时间高压,随后酶解液过两次滤膜,得到粗多肽,粗多肽精制后得到两种较纯的多肽,利用这种技术制备的多肽活性更高、同时能够大幅度缩短生产周期、增加收率。(The invention provides a preparation method of a pilose antler extract, and particularly relates to a preparation method of pilose antler polypeptide, which is characterized in that after fresh pilose antler is treated, homogenization treatment is carried out, alkaline protease is added, enzymolysis is carried out under the action of certain pressure, short-time high pressure is carried out on enzymolysis liquid after the enzymolysis is finished, then the enzymolysis liquid passes through twice filter membranes to obtain crude polypeptide, and two purer polypeptides are obtained after the crude polypeptide is refined.)

1. A preparation method of pilose antler polypeptide is characterized by comprising the following steps:

(1) sample pretreatment: slicing fresh cornu Cervi Pantotrichum, pulverizing, adding anhydrous ethanol at weight ratio of 1:5-10, soaking for 1-2 hr, filtering, and collecting residue;

(2) and (3) mixing the residue in the step (1) according to the ratio of 1: 10-15 adding distilled water, homogenizing by using a homogenizer, adjusting the pH value of the fresh pilose antler slurry to 9-11 after homogenizing, simultaneously adding alkaline protease, then putting the pilose antler enzymatic hydrolysate into an ultrahigh pressure container, wherein the pressure is 50-60Mp, the temperature is 40-50 ℃, the pressure maintaining time is 20-40min, then increasing the equipment pressure to be more than 400 MPa, the pressure maintaining time is 30-120 seconds, decompressing to obtain the slurry after enzymolysis of the fresh pilose antler, and then adjusting the pH value of the enzymolysis solution to be neutral;

(3) centrifuging the enzymolysis liquid in the step (2), collecting supernatant, filtering by using a plate-and-frame filter, passing through a middle control fiber filter membrane with the molecular weight of 10KDa again, passing the filtrate through a middle control fiber filter membrane with the molecular weight of 1000Da, discarding the filtrate with the molecular weight of 1000Da, and freeze-drying the stock solution to obtain crude polypeptide of the pilose antler;

(4) and (4) carrying out sephadex separation on the pilose antler crude polypeptide in the step (3) for 2 times, and freeze-drying to obtain 2 polypeptides with single components, namely APP-1 and APP-2.

2. The method for preparing antler polypeptide according to claim 1, wherein the content of the crude polypeptide in step (3) is 79.24-82.1% and the yield is 1.85-2.44% by detecting with biuret method.

3. The method for preparing the antler polypeptide according to claim 1, wherein the Sephadex gel in step (4) is Sephadex G-75.

4. The method for preparing the antler polypeptide of claim 1, wherein the SDS-PAGE electrophoresis of the antler polypeptide APP-1 and APP-2 in step (4) shows that both have a single band with molecular weights of 2546.2 and 4627.1, respectively.

5. The method for preparing the antler polypeptide according to claim 1, wherein the purity of the antler polypeptide APP-1 and APP-2 in the step (4) is 94.35-95.72% and 96.17-97.25%, respectively, and the yield is 0.55-0.68% and 0.87-0.94%, respectively.

6. The method for preparing the polypeptide of pilose antler of claim 1, wherein the obtained crude polypeptide of pilose antler, polypeptide APP-1 and APP-2 can be used for preparing food and health food for improving blood sugar and immunity.

Technical Field

The invention provides a preparation method of a pilose antler extract, in particular relates to a preparation method of pilose antler polypeptide, and belongs to the technical field of biology.

Background

Cornu Cervi Pantotrichum is an animal belonging to Cervidae of mammalian class of phylum chordata, and is young horn of unossified and dense villus of male deer of Cervus nippon or Cervus elaphus. Velvet antler is recorded in Shen nong Ben Cao Jing, which indicates that velvet antler exclusively enters Mingmen and Du, and enters liver. Sweet and salty in nature, the nature of pure yang, including the qi produced, belong to the middle-grade. Li Shizhen recorded that Tortoise and deer both are effective in prolonging life. The normal inverted tail of the deer nose can dredge the governor vessel, so the horn is used for nourishing the life, replenishing essence and tonifying qi, and it is also used for nourishing yang. It is the physical mystery and mental work. Moreover, modern pharmacological studies also show that the pilose antler has the effects of resisting inflammation, inhibiting and eliminating free radicals, delaying senility, promoting wound healing and the like, the pilose antler polypeptide extracted from the pilose antler can promote the proliferation and differentiation of in-vitro chondrocytes and osteoblasts and inhibit the apoptosis of the chondrocytes, and meanwhile, literature studies also suggest that the liver and kidney meridian entering drugs represented by the pilose antler and the like are used for treating the arthralgia syndrome "

The use frequency of the formula (I) is 39.3%.

The pilose antler contains more complex chemical components, and the types of amino acids are more than 19, including essential amino acids which can not be synthesized by human body, 10 phospholipid components, 9 fatty acids (the contents of oleic acid, linoleic acid and linolenic acid with the strongest biological activity are higher), glycolipids, sugars, sterols, hormone-like substances, prostaglandins, cerebrins, ribonucleic acids, deoxyribonucleic acids, adenosine triphosphate, chondroitin sulfate, polyamines, peptides, lipoproteins, vitamins, enzymes, various trace elements and the like.

The antler polypeptide is a product obtained by hydrolyzing antler protein or a compound with the molecular weight less than 10kDa and directly separated from antler, has various activities, such as biological activities of promoting bone development, regulating immunity, promoting cell proliferation, nerve regeneration and the like, and the main method for preparing the antler polypeptide at present is acidolysis and enzymolysis for preparing the polypeptide.

Disclosure of Invention

The invention aims to provide a technical method for separating and preparing pilose antler polypeptide, which can greatly shorten the time for preparing polypeptide by enzymolysis, improve the activity of the polypeptide, improve the extraction rate of the polypeptide and obtain high-activity polypeptide components. The invention is realized by the following steps:

(1) sample pretreatment: slicing fresh cornu Cervi Pantotrichum, pulverizing, adding anhydrous ethanol at weight ratio of 1:5-10, soaking for 1-2 hr, filtering, and collecting residue.

(2) And (3) mixing the residue in the step (1) according to the ratio of 1: 10-15 adding distilled water, homogenizing with a homogenizer, adjusting pH of the fresh cornu Cervi Pantotrichum slurry to 9-11, and adding alkaline protease. And then putting the antler enzymatic hydrolysate into an ultrahigh pressure container, keeping the pressure at 50-60Mp and the temperature at 40-50 ℃ for 20-40min, then increasing the pressure of the equipment to be more than 400 MPa, keeping the pressure for 30-120 seconds, relieving the pressure to obtain slurry after enzymolysis of the fresh antler, and then adjusting the pH of the enzymatic hydrolysate to be neutral.

(3) Centrifuging the enzymolysis liquid in the step (2), collecting supernatant, filtering by using a plate-and-frame filter, passing through a middle control fiber filter membrane with the molecular weight of 10KDa again, passing the filtrate through a middle control fiber filter membrane with the molecular weight of 1000Da, discarding the filtrate with the molecular weight of 1000Da, and freeze-drying the stock solution to obtain the crude polypeptide of the pilose antler.

(4) And (4) carrying out sephadex separation on the pilose antler crude polypeptide in the step (3) for 2 times, and freeze-drying to obtain 2 polypeptides with single components, namely APP-1 and APP-2.

Further, the content of the crude polypeptide in the step (3) is 79.24-82.1% and the yield is 1.85-2.44% by detecting with a biuret method.

Further, the Sephadex gel in the step (4) is preferably Sephadex G-75.

Further, SDS-PAGE electrophoresis of the antler polypeptide APP-1 and APP-2 in the step (4) shows that the two polypeptides are a single band, and the molecular weights are 2546.2 and 4627.1 respectively.

Further, in the step (4), the purity of the antler polypeptide APP-1 and APP-2 is 94.35-95.72% and 96.17-97.25% respectively, and the yield is 0.55-0.68% and 0.87-0.94% respectively.

The invention has the positive effects that:

1) shortens the enzymolysis time of the polypeptide of the pilose antler, improves the reaction efficiency and reduces the production cost.

2) Increase the product yield and improve the activity of the polypeptide.

3) Obtaining 2 polypeptide components with high purity and single component.

Experimental example 1 preparation of conventional velvet antler polypeptide

(1) Sample pretreatment: slicing fresh cornu Cervi Pantotrichum, pulverizing, adding anhydrous ethanol at weight ratio of 1-10, soaking for 2 hr, filtering, and collecting residue.

(2) And (3) mixing the residue in the step (1) according to the ratio of 1: 15 adding distilled water, homogenizing with a homogenizer, adjusting pH of the fresh cornu Cervi Pantotrichum slurry to 10.5 after homogenizing, simultaneously adding alkaline protease for enzymolysis at 50 deg.C for 7h, inactivating enzyme at 95 deg.C, and adjusting pH of the enzymolysis solution to neutral.

(3) Centrifuging the enzymolysis liquid in the step (2), collecting supernatant, filtering by using a plate-and-frame filter, passing through a middle control fiber filter membrane with the molecular weight of 10KDa again, passing the filtrate through a middle control fiber filter membrane with the molecular weight of 1000Da, discarding the filtrate with the molecular weight of 1000Da, and freeze-drying the stock solution to obtain crude polypeptide of the pilose antler, wherein the content of the polypeptide is 71.53% and the yield is 1.23% through a biuret method.

(4) And (3) separating the crude polypeptide of the pilose antler in the step (3) by sephadex for 2 times, and freeze-drying to obtain 2 polypeptides with single components, namely APP-C1 and APP-C2, wherein the molecular weights of the two polypeptides are 4823.5 and 6138.8 respectively, the purities of the two polypeptides are 89.32% and 85.65%, and the yields of the two polypeptides are 0.12% and 0.26%, respectively.

Experimental example 2 influence of ultrahigh pressure equipment pressure on yield of pilose antler polypeptide

The same material-liquid ratio, reaction temperature and pressure maintaining time are selected in the experiment, and the influence of pressure on the yield of the crude polypeptide of the pilose antler is mainly considered

Experimental example 3 Effect of pilose antler polypeptide on blood glucose and glycated hemoglobin in diabetic mice

Model building

100 ICR mice are adaptively fed for 7 days, 10 mice are randomly selected as a blank control group and fed with normal feed, other 90 mice are fed with high-fat feed for 30 days, the mice are fasted in the evening of the day 30 without water supply, 30 orbital bleeding is randomly extracted on the day 31, serum is obtained by centrifugation, TC and TG are measured and are higher than normal values, and the significant difference exists. Injecting 100mg/kgSTZ citric acid buffer solution into abdominal cavity, feeding high fat feed for 10 days, fasting for no water supply in the evening of 10 days, collecting blood at the tail part of 11 days to measure blood sugar value, measuring fasting blood sugar more than or equal to 16.8mmol/L as success of molding, selecting 60 mice in the molding, randomly dividing into 6 groups, respectively including model group, cornu Cervi Pantotrichum crude polypeptide group, APP-1, APP-2, APP-C1 and APP-C2, each group including 10 mice, feeding gastric physiological saline into blank control group and model group every day, respectively feeding corresponding drugs into other administration groups, and feeding at dosage of 0.1mL/10g for 40 days.

Results of the experiment

Remarking: APP is a high-pressure treatment enzymolysis group, APP-C is a conventional enzymolysis group, # # represents that a model group has significant difference compared with a blank group, # # represents that a model group has significant difference compared with an administration group, and a represents that the APP group has significant difference compared with the APP-C.

Detailed Description